Escolar Documentos
Profissional Documentos
Cultura Documentos
(Received June 2, 2004; revised November 1, 2004; accepted May 14, 2005)
2193
0098-0331/05/0900-2193/0 # 2005 Springer Science + Business Media, Inc.
2194 KONG ET AL.
INTRODUCTION
Sampling Citrus Orchards. Two orchards planted with Citrus sinensis (L.)
Osb. (plants, 2.5 4 m) in Guangzhou Suburb, China (23-06 0 N, 113-18 0 E),
VOLATILE ALLELOCHEMICALS IN THE Ageratum conyzoides 2195
were selected. One orchard had been intercropped with A. conyzoides since
2001. The A. conyzoides plants grew luxuriantly and covered the orchard floor
that was free from other weeds. Another citrus orchard without A. conyzoides
served as the control. The major weeds in this orchard were Bidens pilosa,
Digitaria sanguinalis, and Cyperus difformis. If any A. conyzoides appeared in
the control site, they were hand-weeded since October 2002. The experimental
orchards were in a subtropical climate zone, with a mean annual temperature of
21.8-C and a mean annual rainfall of 1682 mm. There were no significant
differences in pH, organic matter content, and fertility status between the
intercropped and control site soils (pH 5.6; organic matter 19.7 g kgj1; total
N 0.97 g kgj1; available N 40.8 mg kgj1; total P 0.48 g kgj1; available P 13.8
mg kgj1; total K 102.9 g kgj1; available K 98.7 mg kgj1).
Population Densities of Mites. A. newsami and P. citri were used because
A. newsami is the most common Amblyseius spp. predatory mite and P. citri is a
major citrus arthropod pest in citrus orchards in South China. Fifteen citrus
plants, whose canopies did not touch each other, were randomly selected from
two orchards, respectively. For the population census, 50 representative spatial
leaves of each citrus plant were selected, and adult mites were counted twice a
day (10: 00 a.m. and 4:00 p.m.) for three consecutive days (Tao and Luo, 1992).
Data were subjected to ANOVA with Duncans multiple range test. The mean
number of individual mites (A. newsami or P. citri) per leaf indicated their
population densities in the orchards.
Effect of the essential oil on the mites was studied in a nonintercropped
citrus orchard (i.e., control without A. conyzoides). Twenty-seven citrus plants
(3 9), whose tree canopies were separated from one another by at least 15 m,
were randomly selected. Then, a 5% essential oil emulsion (10 kg) of
A. conyzoides was sprayed on 18 citrus plants (2 9). Ten kg water served
as a control (1 9 citrus plants). After 24 and 48 hr, the population densities
(mean individuals per leaf ) of adult mite A. newsami and P. citri on each of 50
representative spatial leaves of each plant were counted as described above.
Experiments were carried out in June 2003, when A. conyzoides was at its
peak flowering stage, and mites A. newsami and P. citri were active and
abundant. Sampling dates were cloudy with a 27 T 5-C air temperature and 70 T
10% relative humidity. Pesticides have never been applied in either citrus
orchard since October 2002.
Collection and Analysis of Volatiles. Collection and analysis of volatiles in
the orchards was as follows (Kong et al., 2004a). Nine sampling locations were
randomly selected from both citrus orchards. Air samples 1 m above the ground
at 2 9 locations each were collected by Timing Minipump air samplers with a
flow meter TMP-1500 (China Electrical Instruments Co.) and pumped through
the sampling tubes (Tekmar Co., USA) packed with Tenax and Carbonsieve at a
flow rate of 0.5 l/min for 30 min. The volatile chemicals in air samplers were
2196 KONG ET AL.
adsorbed onto the solid adsorbents, and sampling tubes were installed into a
Tekmar 6032 Aero-trap Autosampler for thermal desorbing. The tubes were
rapidly heated to 225-C, and their volatiles were desorbed and carried by
helium to a Tekmar 3000 Purge & Trap Concentrator where they were trapped
again. The concentrator was heated at a rate of more than 400-C/min, and the
trapped volatiles were transported to a Hewlett-Packard P 5972 GC/MSD with
an HP-5 bonded stage fused-silica capillary column (30 m 2.5 mm) for
analysis. The initial oven temperature (70-C) was maintained for 2 min and then
increased to 180-C at a rate of 10-C/min. The column was maintained at 180-C
for 5 min, and then the oven temperature was raised to 250-C at 15-C/min.
Mass spectra were repetitively scanned from 35 to 450 amu every 2 sec.
Ionization was selected in the electron impact mode (EI) at 70 eV. The chemical
constituents were identified by peak matching against standards in the NIST 95
Computer Library or by spectral similarity to an authentic reference compound
(Aldrich Chemical Co.). The relative amounts of the chemical constituents were
calculated by integrating all peaks with an area greater than 0.1%.
Collection and analysis of volatiles from fresh leaves and the essential oil
of A. conyzoides were performed using the technique of multiple headspace
solid-phase microextraction (Augusto et al., 2003; Ezquerro et al., 2003). GC/
MSD and manipulation was the same as described above.
The essential oil was obtained by placing fresh leaves of A. conyzoides into
a desiccator with a liquid nitrogen cold trap and pumped into a vacuum (Kong
et al., 2002). It was then subjected to silica gel column chromatography with n-
hexane/ether (9:1 and then 4:6; v/v) mixture. It produced five major
components, ageratochromene, demethoxy-ageratochromene, b-caryophyllene,
E-b-farnesene, and a-bisabolene.
Olfactory Responses of Mites to Volatiles. Adult mites of both A. newsami
and P. citri were collected from citrus orchards and reared in a petri dish (9-cm
diam.; 1.8-cm depth) at 25-C and 50% relative humidity. The dish was placed in
a transparent container over a pad of black colored velvet and a sponge (8-cm
diam.; 1 cm thick). Because the mites are translucent, the black background
enhanced visual detection. The sponge was allowed to imbibe and maintain
water by partially adding water daily.
The olfactory response of the mites to volatiles was studied with a slightly
altered airflow four-armed olfactometer designed by Pettersson (1970). The size
of the exposure chamber was halved (ray of crescents: 67.5 mm, inner height: 5
mm, inner diameter of stainless tubes: 3 mm). A flow rate of 300 ml/min
through each arm was maintained to create odor fields with sharp boundaries.
The exposure chamber and TV monitor screen (21 00 ) were interfaced with a
multifunctional MC-1 camera (China Optical Co.) with the following features:
sensitive element, 1/3 00 , 500(H) 582(V) lines; resolution, 420 lines; optical
attachment, eyepiece adapter (39-mm diam for stereomicroscope and 23.3-mm
VOLATILE ALLELOCHEMICALS IN THE Ageratum conyzoides 2197
RESULTS
FIG. 1. Population density (mean individuals per leaf ) of mites on citrus trees (A, in the
A. conyzoides intercropped citrus orchard; B, in the A. conyzoides nonintercropped citrus
orchard; C, in the A. conyzoides nonintercropped citrus orchard, after 24 hr of spraying
5% A. conyzoides essential oil emulsion; D, in the A. conyzoides nonintercropped citrus
orchard, after 48 hr of spraying 5% A. conyzoides essential oil emulsion).
nonintercropped citrus orchards, but that their population densities had obvious
differences. In the A. conyzoides intercropped orchard, the population density of
A. newsami reached 0.3 individual per leaf, and the P. citri population was
markedly decreased. In the A. conyzoides nonintercropped orchard, the A.
newsami population was below 0.1 individual per leaf, and the P. citri
population was increased over 0.2 individual per leaf. This indicated that
intercropping with A. conyzoides in citrus orchards increased the A. newsami
population and reduced the population density of citrus red mite to low and
noninjurious levels. This was possibly correlated with volatiles in the air.
Volatile Allelochemicals and Their Effects on Mites. Several terpenes, such
as carene, cubebene, copaene, limonene, myrcene, and pinene, were detected in
the air in both the A. conyzoides intercropped and nonintercropped orchards.
Abundant ageratochromene, demethoxy-ageratochromene, a-bisabolene, b-
caryophyllene, and E-b-farnesene were found in samples from the A. conyzoides
intercropped orchard, fresh leaves, and the essential oil of A. conyzoides, but
these were not detected in the air samples above the A. conyzoides noninter-
cropped orchard (Table 1). These volatile components in the orchards might
come from understory plants, soil, or other organisms, but ageratochromene,
demethoxy-ageratochromene, b-caryophyllene, a-bisabolene, and E-b-farnesene
were all major components of A. conyzoides fresh leaves and essential oil.
Particularly, ageratochromene and demethoxy-ageratochromene are specific in
the Ageratum genus (Kong et al., 2002, 2004a; Okunade, 2002). These
VOLATILE ALLELOCHEMICALS IN THE Ageratum conyzoides 2199
Data were the relative amounts (%) of constituents from each sample.
* Means T SE from three independent experiments with nine sampling locations in the A. conyzoides
intercropped or nonintercropped citrus orchards for each determination are shown. Data not
followed by the same letter in a line are significantly different at P < 0.05, with Duncans multiple
range test.
chemicals did not occur in the air samples of the citrus orchard without
A. conyzoides, indicating that they were primarily released from A. conyzoides.
The presence of these volatile allelochemicals thus may make a contribution
toward regulating the population of the mites in the A. conyzoides intercropped
orchard.
Olfactometer trials showed that A. conyzoides fresh leaves, the essential
oil, and its four components had different effects on the olfactory responses of
A. newsami and P. citri (Table 2). The fresh leaves and essential oil as well as
demethoxy-ageratochromene, b-caryophyllene, a-bisabolene, and E-b-farnesene
strongly attracted A. newsami and slightly repelled P. citri at the test tem-
perature (25-C). It is noted that an olfactory response of mites to ageratochro-
mene was not achieved because ageratochromene is a crystal (mp 46Y47-C) and
difficult to volatilize at this test temperature. The olfactory response of
A. newsami to each individual component was clearly lower than to those of
A. conyzoides fresh leaves and the essential oil (Table 2), suggesting that this
effect could be intensified with the combinations of several components from
A. conyzoides.
Field experiments (Figure 1) showed that spraying the essential oil of
A. conyzoides on the citrus trees in the A. conyzoides nonintercropped citrus
2200 KONG ET AL.
Data were mean value of three independent experiments with 50 mites for each determination and
not followed by the same letter in a column are significantly different at P < 0.05, with Duncans
multiple range test.
DISCUSSION
The role of orchard ground cover plants in improving habitat and enriching
natural enemies of pests has been known for a long time (Bugg et al., 1990; Fye,
1983; House and Alzugaray, 1989; Lawton, 1982). It is necessary to select
beneficial plant species and pay attention to their chemical interactions with
understory plants and other organisms in the orchards. A citrus orchard is a
relative stable and complicated ecosystem. The population dynamics of mites in
citrus orchards often depends on the associated organisms, atmospheric tem-
perature, precipitation, and other factors (Liang and Huang, 1994). In this study,
A. conyzoides produced and released volatile allelochemicals into the air. These
were at least, in part, responsible for the population variations of mites,
VOLATILE ALLELOCHEMICALS IN THE Ageratum conyzoides 2201
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